WO2001094918A1 - Reference device for evaluating the performance of a confocal laser scanning microscope, and a method and system for performing that evaluation - Google Patents
Reference device for evaluating the performance of a confocal laser scanning microscope, and a method and system for performing that evaluation Download PDFInfo
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- WO2001094918A1 WO2001094918A1 PCT/EP2001/006365 EP0106365W WO0194918A1 WO 2001094918 A1 WO2001094918 A1 WO 2001094918A1 EP 0106365 W EP0106365 W EP 0106365W WO 0194918 A1 WO0194918 A1 WO 0194918A1
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Classifications
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration
- G01N21/274—Calibration, base line adjustment, drift correction
- G01N21/278—Constitution of standards
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
- G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
Definitions
- the invention concerns a reference device for evaluating the performance of a confocal laser scan microscope of the kind
- a DNA binding array or the like e.g. a DNA binding array of the type described in U.S. Patent No.5, 143 , 854.
- the invention also concerns a method for evaluating the performance of a confocal laser scan microscope of the above mentioned kind.
- the invention more in particular concerns an evaluation method enabling the characterization of a confocal laser scan microscope of the above mentioned kind in terms of quantitative signal detection sensitivity, limit of detection, uniformity of the confocal volume over the scan 25 field of view, spatial resolution of the scanning process and dynamic behavior of the measured signal over the scan field of view, said measured signal corresponding to the fluorescent light received.
- the invention further concerns a system for evaluating the performance of a confocal laser scan microscope which is apt to be used for performing a two dimensional quantitative fluorescence measurement of test matter distributed on a flat surface of a substrate. 35 Background
- Figure 1 shows the optical setup of a 2-D flying spot confocal laser scan microscope, using for fluorescence excitation a laser beam 11, a dichroic beam splitter 12, a 2 -dimensional scan engine 13 for spatial beam deflection in two orthogonal directions (X- Y) and a lens 14 for focusing the laser beam into an object plane 15.
- Fluorescent light of a longer wavelength than the excitation laser 11 is generated by exciting fluorescent molecules in the object plane 15.
- Fluorescent light emitted by fluorophores located in the object plane 15 of the scanned area is collected by lens 14 and then transmitted by means of the scan engine 13 and the dichroic beam splitter 12 as a fluorescent light beam 17 which is focused by lens 18 into a pinhole aperture 19 in a conjugate plane 21 in front of a photodetection device 22.
- confocal imaging which is currently used to discriminate the generally weak fluorescence signal from background radiation, is illustrated in Fig. 2. Only optical radiation from within the confocal volume Vc, i.e., the fluorescence signal, is detected by the photodetector 22. Vc is defined by the optical transfer function of the detection optics (OTFem) and the size of the detector pinhole 19 in the conjugate plane 21. Higher background suppression rates result for smaller confocal volumes Vc .
- the size of the scan field of view is typically in the order of 20 x 20 square millimeter.
- the pixel size of the scan engine 13 for scanning the laser beam 11 in the field of view is typically 1 to 20 micrometer.
- DNA binding arrays consist of a glass chip carrying a chemical system subdivided in adjacent cells, commonly called features.
- the features are characterized by specific probes.
- Specific nucleic acid sequences are immobilized
- the amount of captured nucleic acid on individual features is detected using quantitative fluorescence measurement (the fluorescent dye emits light when excited by light energy of a given wavelength) by sequential pixel reading (scanning) of the features.
- the features are spatially over-sampled by the scanning procedure. (i.e. number of pixels > number of features) for accurate spatial referencing of the glass chip by numerical data analysis and for increased feature signal quality by averaging physically measured light intensities.
- Typical pixel sizes are in the order of 1 to 20 micrometer.
- the x-y position dependence is mainly due to mechanical misalignment and imperfections of optical and opto-mechanical components, such as e.g. the scan engine used for scanning.
- Fig. 4 schematically shows the scanned image of a DNA binding array, e.g. of the type described in U.S. Patent No.5, 143 , 854, which array has a chess-board pattern.
- the scanned image has a lower signal level in the top right corner, due to either inhomogeneous fluorophore density in the scanned object or inhomogeneous sensitivity of the confocal laser scan microscope over the scan field of view.
- the aim of the invention is therefore to provide a reference device, a method and a system of the above mentioned kinds that make it possible to evaluate quantitatively the performance of a confocal laser scan microscope for performing two-dimensional, quantitative fluorescence measurements .
- the main advantages attained with a reference device, method, and system according to the invention are that they allow a quantitative and highly accurate evaluation of the performance of a confocal laser microscope for scanning DNA binding arrays of the above mentioned kind, and that this evaluation makes it possible to evaluate quantitatively measurement results obtained by scanning with such a microscope, e.g. a sample DNA binding array to be analyzed.
- the evaluation performed according to the invention includes the measurement of the following characteristics: a) quantitative signal detection sensitivity, b) quantitative signal detection limit c) uniformity of the confocal volume over the scan field of view, d) spatial resolution of the scanning process, and e) dynamic behavior of the measured signal over the scan field of view, said measured signal corresponding to the fluorescent light received.
- Fig. 1 shows a schematic representation of the basic setup of a confocal laser scan microscope for performing a two-dimensional, quantitative fluorescence measurement
- Fig. 2 schematically shows a confocal volume Vc in an object plane
- Figures 3a, 3b and 3c show schematic representations of various forms of non-uniformity of the scanned confocal volume over the scan field of view
- Fig. 4 shows a schematic representation of a scanned image of a DNA binding array
- Fig. 5a shows a top view of a first embodiment of a reference device according to the invention
- Fig. 5b shows a cross-section through a plane A-A of the embodiment shown by Fig. 5a
- Fig. 6 shows the shape of a representative electrical signal obtained by measuring fluorescent light emitted by fluorescent zones located in a row of the array represented in Figures 5a and 5b.
- Fig. 7a shows a top view of a second embodiment of a reference device according to the invention
- Fig. 7b shows a cross-section of the embodiment shown by Fig. 7a.
- Fig. 1 schematically shows a basic setup of a confocal laser scan microscope for two-dimensional, quantitative fluorescence measurement in case of a two-dimensional flying spot.
- An excitation laser beam 11, which is transmitted through a dichroic beam splitter 12, is spatially scanned by means of a two-axis scan engine 13, e.g., a galvo-scanner, in two axis X, Y, perpendicular to each other, and is focused by a lens 14 into an object plane 15 which is parallel to a X-Y-plane defined by the axis X and Y, and which is perpendicular to a third axis Z which is perpendicular to the X-Y-plane.
- a two-axis scan engine 13 e.g., a galvo-scanner
- Fluorophores within the confocal volume Vc in the object plane 15 are excited by the focused laser spot 16 and the fluorescent light 17 generated by excitation of those fluorophores is collected and imaged by a lens 18 into a detector pinhole 19 in the conjugate plane 21 and detected by photodetector 22.
- Confocal volume Vc is ideally a cylindrical volume having a rotation axis parallel to the Z axis and a circular cross-section.
- the confocal volume Vc is defined by the optical transfer function (OTFem) of the detection optics and the size and the shape of the detector pinhole 19 in the conjugate plane 21.
- OTFem optical transfer function
- Only optical radiation from within the confocal volume Vc is detected by the photodetector 22.
- the concept of confocal imaging allows high background suppression rates for detecting weak signal levels, as is commonly the case in fluorescence measurements.
- Figures 3a, 3b and 3c show schematic representations in the plane Y-Z of various forms of non-uniformity of the scanned confocal volume Vc, that is of deviations of the shape of this volume from the ideal shape represented in Fig. 2. These deviations cause inhomogenities of the amplitude of the fluorescent light intensity signal measured over the scanned area.
- Fig. 3a shows a scanned confocal volume 24 which is tilted with respect to an ideal or nominal confocal volume 23.
- Fig. 3b shows a scanned confocal volume 25 which has not a constant width and which is thus non-uniform compared with the nominal confocal volume 23.
- Fig. 3c shows a scanned confocal volume 26 having a shape which is distorted with respect to the nominal confocal volume 23.
- Fig. 4 shows a schematic representation of a scanned image of DNA binding array 31 of the type described in U.S. Patent No.5, 143 , 854.
- Array 31 has a chess-board array of fluorescent points 32 apt to emit fluorescence light when it is irradiated with excitation light.
- the deviation of the signal intensity of each pixel from a predetermined value which is given e.g. by the signal intensity averaged over the whole scanned image, gives a quantitative parameter for the performance of a confocal laser scan microscope.
- the number of fluorophores per area can be evaluated from the concentration of dissolved fluorophores. Therefore, the signal intensity measured and the measurement sensitivity are related to the number of fluorophores per area in a quantitative manner.
- Fig. 7a shows a top view and Fig. 7b a cross-section of a second embodiment of a reference device according to the invention for a quantitative evaluation of the homogeneity and spatial resolution of a confocal laser scan microscope.
- the reference device 51 shown by Figures 7a and 7b consists of a top glass plate 52 on a glass substrate 53 having an area of 16 x 16 square millimeter.
- Cavity 54 is filled with uniformly dissolved fluorophores having a predetermined concentration, which leads to a predetermined spatial distribution of fluorescent zones over the area of cavity 54. This spatial distribution is not determined by the uniformly dissolved fluorophores themselves, but by the structure of cavity 5 .
- Cavity 54 is covered by the glass top plate 52 which has a lower outer surface.
- the latter space is at least partially filled with dissolved fluorophores .
- Glass substrate 53 has e.g. identical dimensions and preferably the same optical properties as the substrate of DNA binding array 31 described above with reference to Fig.
- Glass cover 52 of reference device 51 has identical optical properties as a glass substrate of a given DNA binding array 31 of the kind described above with reference to Fig. 4. Reference device 51 can therefore be scanned by a confocal laser scan microscope under the same optical conditions.
- the microstructured cavity 54 comprises different patterns of fluorescent zones.
- each non-fluorescent zone is represented by a shaded surface.
- a first pattern of fluorescent zones comprises just two non- fluorescent zones each represented in Fig. 7 by a shaded square.
- fluorescent zones having this first pattern are located at each of the corner zones 55, 56, 57, 58 of reference device 51.
- the measured signals corresponding to the intensity of fluorescent light emitted from these corner zones are evaluated in order to assess the degree of uniformity over the scan field of view of the scanning performed with a confocal laser scan microscope.
- LO > to to l- 1 H 1 in o in o in O in
- the above mentioned use of the invention for evaluating the performance of a confocal laser scan microscope substantially comprises scanning a reference device according to the invention with a microscope to be evaluated in order to obtain a first set of measurement values, processing said first set of measurement values in order to obtain correction factors, storing said correction factors, scanning a sample, e.g. a DNA binding array, with the evaluated microscope in order to obtain a second set of measurement values, and correcting said second set of measurement values with said correction factors in order to obtain a third set of values which are free from deviations due to the performance of the scanner and which therefore more accurately correspond to characteristics of the particular sample examined.
Abstract
Description
Claims
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002409353A CA2409353C (en) | 2000-06-07 | 2001-06-05 | Reference device for evaluating the performance of a confocal laser scanning microscope, and a method and system for performing that evaluation |
EP01947341A EP1287339B1 (en) | 2000-06-07 | 2001-06-05 | Reference device for evaluating the performance of a confocal laser scanning microscope, and a method and system for performing that evaluation |
JP2002502419A JP3706367B2 (en) | 2000-06-07 | 2001-06-05 | Reference device for evaluating the performance of a confocal laser scanning microscope, and method and system for performing the evaluation |
DE60130452T DE60130452T2 (en) | 2000-06-07 | 2001-06-05 | Reference device for evaluating the function of a confocal laser scanning microscope, and method and system for carrying out the evaluation |
US10/297,631 US7053384B2 (en) | 2000-06-07 | 2001-06-05 | Reference device for evaluating the performance of a confocal laser scanning microscope, and a method and system for performing that evaluation |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP00810496.0 | 2000-06-07 | ||
EP00810496A EP1162450A1 (en) | 2000-06-07 | 2000-06-07 | Reference device for evaluating the performance of a confocal laser scanning microscope, and a method and system for performing that evaluation |
Publications (1)
Publication Number | Publication Date |
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WO2001094918A1 true WO2001094918A1 (en) | 2001-12-13 |
Family
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Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP2001/006365 WO2001094918A1 (en) | 2000-06-07 | 2001-06-05 | Reference device for evaluating the performance of a confocal laser scanning microscope, and a method and system for performing that evaluation |
Country Status (8)
Country | Link |
---|---|
US (1) | US7053384B2 (en) |
EP (2) | EP1162450A1 (en) |
JP (1) | JP3706367B2 (en) |
AT (1) | ATE373228T1 (en) |
CA (1) | CA2409353C (en) |
DE (1) | DE60130452T2 (en) |
ES (1) | ES2291328T3 (en) |
WO (1) | WO2001094918A1 (en) |
Families Citing this family (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE10100247A1 (en) | 2001-01-05 | 2002-07-11 | Leica Microsystems | Interference microscope and method for operating an interference microscope |
DE10100246A1 (en) * | 2001-01-05 | 2002-07-11 | Leica Microsystems | Confocal or double confocal microscope especially for imaging biomedical objects has a reference object integral with an object supporting carrier that can be used for rapid alignment and refocusing of the microscope |
AU2002247765B2 (en) * | 2001-03-28 | 2007-04-26 | Clondiag Chip Technologies Gmbh | Device for referencing fluorescence signals |
US20050030601A1 (en) * | 2003-06-12 | 2005-02-10 | Affymetrix, Inc. | System and method for scanner instrument calibration using a calibration standard |
DE102005049364B4 (en) * | 2005-03-18 | 2023-05-25 | BAM Bundesanstalt für Materialforschung und -prüfung | Multifunctional calibration device and kit and their uses for characterizing luminescence measurement systems |
CH708797B1 (en) * | 2007-08-17 | 2015-05-15 | Tecan Trading Ag | Sample part magazine for a slide transport device of a laser scanner device. |
DE102008007178A1 (en) | 2008-01-30 | 2009-08-06 | Carl Zeiss Microimaging Gmbh | Calibration device and laser scanning microscope with such a calibration device |
TWI421720B (en) * | 2010-09-29 | 2014-01-01 | Univ Nat Sun Yat Sen | A performance evaluation apparatus for an electromagnetic stirrer and a method thereof |
US9864190B2 (en) * | 2011-02-24 | 2018-01-09 | The Board Of Trustees Of The Leland Stanford Junior University | Confocal microscope, system and method therefor |
JP6363991B2 (en) * | 2013-03-13 | 2018-07-25 | オリンパス株式会社 | Evaluation method of optical analyzer |
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-
2000
- 2000-06-07 EP EP00810496A patent/EP1162450A1/en not_active Withdrawn
-
2001
- 2001-06-05 EP EP01947341A patent/EP1287339B1/en not_active Expired - Lifetime
- 2001-06-05 ES ES01947341T patent/ES2291328T3/en not_active Expired - Lifetime
- 2001-06-05 US US10/297,631 patent/US7053384B2/en not_active Expired - Fee Related
- 2001-06-05 CA CA002409353A patent/CA2409353C/en not_active Expired - Fee Related
- 2001-06-05 DE DE60130452T patent/DE60130452T2/en not_active Expired - Lifetime
- 2001-06-05 JP JP2002502419A patent/JP3706367B2/en not_active Expired - Fee Related
- 2001-06-05 WO PCT/EP2001/006365 patent/WO2001094918A1/en active IP Right Grant
- 2001-06-05 AT AT01947341T patent/ATE373228T1/en active
Patent Citations (10)
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JPS5663242A (en) * | 1979-10-30 | 1981-05-29 | Yokogawa Hokushin Electric Corp | Standard sample for moisture meter by infrared ray |
US4302678A (en) * | 1980-01-25 | 1981-11-24 | Magnaflux Corporation | Fluorescent standard for scanning devices |
DE3148912A1 (en) * | 1981-12-10 | 1983-06-23 | Wolfgang Dr. 6301 Pohlheim Oberheim | Mercury reference standard |
US4662745A (en) * | 1986-02-05 | 1987-05-05 | Atlantic Richfield Company | Reflectance and luminescence calibration plate having a near-Lambertian surface and method for making the same |
US5143854A (en) * | 1989-06-07 | 1992-09-01 | Affymax Technologies N.V. | Large scale photolithographic solid phase synthesis of polypeptides and receptor binding screening thereof |
US5414258A (en) * | 1993-11-22 | 1995-05-09 | Angstrom Technologies, Inc. | Apparatus and method for calibration of fluorescence detectors |
US5689110A (en) * | 1994-09-02 | 1997-11-18 | Biometric Imaging, Inc. | Calibration method and apparatus for optical scanner |
US5581631A (en) * | 1994-09-20 | 1996-12-03 | Neopath, Inc. | Cytological system image collection integrity checking apparatus |
WO1998028592A1 (en) * | 1996-12-23 | 1998-07-02 | Ruprecht-Karls-Universität Heidelberg | Method and devices for measuring distances between object structures |
US5838435A (en) * | 1997-10-20 | 1998-11-17 | Sandia Corporation | Calibration method for spectroscopic systems |
Also Published As
Publication number | Publication date |
---|---|
DE60130452D1 (en) | 2007-10-25 |
ES2291328T3 (en) | 2008-03-01 |
US7053384B2 (en) | 2006-05-30 |
CA2409353C (en) | 2009-12-29 |
EP1162450A1 (en) | 2001-12-12 |
ATE373228T1 (en) | 2007-09-15 |
JP3706367B2 (en) | 2005-10-12 |
DE60130452T2 (en) | 2008-06-12 |
JP2003536067A (en) | 2003-12-02 |
EP1287339A1 (en) | 2003-03-05 |
US20040051050A1 (en) | 2004-03-18 |
CA2409353A1 (en) | 2001-12-13 |
EP1287339B1 (en) | 2007-09-12 |
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